Name: personalized needles for microinjections in the rodent brain
Uploaded: 2018-01-24T12:30:00
Description: Watch this Scientific Journal Video about Personalized Needles for Microinjections in the Rodent Brain at JoVE.com

This work has been supported by a grant from the European Community [FP7-PEOPLE-2011-IAPP project 285827 (EPIXCHANGE)].

All experimental protocols were approved by the University of Ferrara Ethical Committee for Animal Experimentation and by the Italian Ministry of Health. The ARRIVE (Animal Research: Reporting In Vivo Experiments11) guidelines have been followed.
1. Preparation of Quartz Needles
<ol>
	<li>Clean and sterilize the quartz capillaries (see Table of Materials) by placing them for 5 min in distilled water, 5 min in ethanol 99%, and 5 min in diethyl-ether.</...

<ol>
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The technique described in this article fulfills the needs outlined in the Introduction for optimizing microinjections that are performed for various purposes12. The needles described here reduce damage to a minimum, essentially non-detectable level; at variance with glass needles (that are prone to bend), quartz needles are rigid and ensure a reliable hit of the desired brain region even in deep coordinates. Moreover, the side hole ensures delivery of the solution even if the tip...

Microinjections have been used for a long time for the delivery of pharmacologically active compounds to modulate neuronal activity in specific brain areas. In addition, they have been used to inject toxins near particular neuronal populations, to mimic neurodegenerative events characteristic of certain diseases, for example 6-hydroxy-dopamine in the nigrostriatal dopamine system to mimic Parkinson&#39;s disease1,2 or the immunotoxin 192 IgG saporin to lesion the cholinergic system3. More recently, microinjection procedures have been used to deliver viral vectors or cells grafts for gene or cell therapy of experimental brain disorders4,5.
The classical type of needles employed in these studies is made of stainless steel. Although easy and practical to use, steel needles have a number of problems6: they are relatively large and may cause tissue damage, with leakage of the blood-brain barrier and activation of astrocytes; moreover, they may produce coring of brain tissue that gets into the needle creating an obstacle or even completely avoiding flow of the desired solution. More recently, glass needles prepared ad hoc from capillaries have been introduced in use7,8. These do not cause significant tissue damage nor astrocyte activation, but are relatively flexible and may bend when introduced in deep structures, reducing the accuracy of localization (personal observations).
There is therefore a need to reduce as much as possible damage (especially when performing experiments to heal damage) while increasing accuracy and reproducibility (i.e., ensure that all solution is delivered and ensure correct localization). Moreover, it would be desirable to use different needle designs to ensure optimal distribution of the injected solution in brain areas with varying geometries. In this article, we describe a protocol to prepare and use quartz needles for microinjections in the rodent brain. Due to the high melting point, quartz capillaries cannot be pulled on a conventional puller and therefore, have not been used in the past to generate needles. Quartz, however, offers some important advantages over glass, in particular high rigidity and break resistance9. Because of their rigidity, quartz needles are ideally suited for injections into ventral brain regions. Because of their high resistance to breakage they can be modeled to include multiple holes, obtaining designs that may prove most effective even when targeting brain regions with complex geometries10.

<table>
	<tbody>
		<tr>
			<td>Quartz capillaries</td>
			<td>Sutter Instruments, Novato, CA USA</td>
			<td>Q100-50-10</td>
			<td>Without filament</td>
		</tr>
		<tr>
			<td>Puller</td>
			<td>Sutter</td>
			<td>P2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette storage jar</td>
			<td>World Precision Instruments (WPI), Sarasota, FL, USA</td>
			<td>E210</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser microdissector</td>
			<td>Leica Microsystems, Wetzlar, Germany</td>
			<td>LMD6500</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton syringe</td>
			<td>Hamilton ILS Innovative Labor Systeme GmbH, St&#376;tzerbach, Germany</td>
			<td>19138-U</td>
			<td></td>
		</tr>
		<tr>
			<td>Microinfusion pump</td>
			<td>Univentor, Zejtun, Malta</td>
			<td>Model 864</td>
			<td></td>
		</tr>
		<tr>
			<td>Manual microinjection pump kit</td>
			<td>WPI</td>
			<td>Item#: MMP-KIT</td>
			<td>Kit allowing for micropipettes to be securely mounted to the stereotactic frame</td>
		</tr>
		<tr>
			<td>Precision Drill</td>
			<td>Proxxon</td>
			<td>28510 MicroMot 50/E</td>
			<td>Ball bearing drive shaft with variable speed</td>
		</tr>
		<tr>
			<td>Artficial Cerebral Spinal Fluid</td>
			<td>Tocris</td>
			<td>3525</td>
			<td></td>
		</tr>
		<tr>
			<td>Needles 26 G Blunt and 30 G Bevel</td>
			<td>Hamilton</td>
			<td>26 G Blunt: 19138-U 
			30 G Bevel: 20757</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>Leica, Germany</td>
			<td>LEICA RM212RT</td>
			<td></td>
		</tr>
	</tbody>
</table>

personalized needles for microinjections in the rodent brain

Microinjections have been used for a long time for the delivery of drugs or toxins within specific brain areas and, more recently, they have been used to deliver gene or cell therapy products. Unfortunately, current microinjection techniques use steel or glass needles that are suboptimal for multiple reasons: in particular, steel needles may cause tissue damage, and glass needles may bend when lowered deeply into the brain, missing the target region. In this article, we describe a protocol to prepare and use quartz needles that combine a number of useful features. These needles do not produce detectable tissue damage and, being very rigid, ensure reliable delivery in the desired brain region even when using deep coordinates. Moreover, it is possible to personalize the design of the needle by making multiple holes of the desired diameter. Multiple holes facilitate the injection of large amounts of solution within a larger area, whereas large holes facilitate the injection of cells. In addition, these quartz needles can be cleaned and re-used, such that the procedure becomes cost-effective.

We compared damage induced by direct microinjection in the rat dorsal hippocampus and striatum using a quartz needle (60 &#181;M external diameter tip; one 20 &#181;M diameter side hole; type C, Figure 1) compared with two classic, 26 G blunt end and 30 G bevel edge stainless steel needles. To this aim, we injected 2 &#181;L of aCSF in the right and left dorsal hippocampus and striatum using respectively the quartz and the steel needle and, 48 h after injecti...

Watch this Scientific Journal Video about Personalized Needles for Microinjections in the Rodent Brain at JoVE.com

Personalized Needles for Microinjections in the Rodent Brain

We describe here a protocol for microinjection in the rodent brain that uses quartz needles. These needles do not produce detectable tissue damage and ensure reliable delivery even in deep regions. Moreover, they can be adapted to research needs by personalized designs and can be re-used.

Microinjections have been used for a long time for the delivery of drugs or toxins within specific brain areas and, more recently, they have been used to ...

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